Using a laser scanning microscope (LSM) selected points (well defined volume units) of the tested material are irradiated by a focused laser beam in response of which information on the intensity of the transmitted, reflected or emitted light is obtained which can be stored generally in digital form. The signal of the laser scanning microscope during scanning a field of predetermined width and length is used to obtain a picture of high resolution for detailed analysis. The picture quality may be further increased by using the LSM in confocal mode, thereby substantially excluding the disturbing effect of the light emanating from points other than the focus point. In most of the commercially available laser scanning microscopes (such as in Zeiss 410 or 510) the confocal mode is a basic feature but it may be used only for the reflected or emitted light (fluorescence). The confocal mode of the LSM provides for the non-destructive optical slicing of the sample and the reconstruction of three dimensional “images”. Highly improved picture quality may be obtained by using a two or more photon laser excitation method which may be strictly limited to the tested area and thereby the disturbing effect of the background radiation (intensity) may be practically completely eliminated (A. Diaspro and M. Robello: Multi-Photon Excitation Microscopy to Study Biosystems, European Microscopy and Analysis, March 1999).
Laser scanning microscopes—when compared to the conventional microscopes and methods—provide a high quality and high resolution information of the sample structure. Nevertheless these methods do not provide any information on the anisotropy and many other physical interactions of the sample that may only be examined with polarization spectroscopy methods.
The use of polarized light provides images of the sample comprising information on the anisotropic structure, e.g. the spatial arrangement of the transition dipoles, and the physical interaction between each other and the micro-environment. The anisotropic properties of the materials influence generally the polarization properties of the light emitted, reflected or transmitted by the materials in an anisotropic way, therefore the examination of the polarization properties of the light emitted, reflected or transmitted by the materials enables conclusions relating to the optical anisotropy and also the molecular order of the tested material. Measurements carried out with polarized light (LD: linear dichroism, CD: circular dichroism) are described by T. C. Oakberg in Application note, Stokes Polarimetry, Hinds Instruments Inc., 1991 news. Similar method may be used for measuring the birefringence, too. The linear polarization of luminescence emission provides important information on the anisotropy of the emission dipoles, therefore the anisotropy value (r) characteristic for this provides an important information on the material structure not obtained by other techniques. The circular polarized luminescence (CPL) content of the emission (emitted light) provides important information on the chiral structure of the material when excited which may not be obtained in any other way. Further important information is the degree of polarization (P) of the fluorescence which allows conclusion on energy transfer between the dipoles, the microviscosity of the surrounding of the molecule, lifetime of an excitation and other important parameters. The definition, measurement and physical content of P, r and CPL is specified in detail by J. R. Lakovicz in his book “Principles of Fluorescence Spectroscopy” and I. Z. Steinberg in his report published in Methods in Enzymology.
During differential polarization imaging as described in detail by Kim et al. in a report published in Biophysical Journal two different images are produced of the sample using orthogonally polarized light, the intensity normalized difference of which provides information on the anisotropic structure of the material or sample. The CD, LD and other differential polarization values of transmitted light provide important information on the anisotropic structure of the material not available with other techniques.
The polarization properties of the fluorescence (emitted light) may be determined by placing a polarizer component (e.g. a polarizer filter) in front of the detector of the LSM, rotating the polarizer filter between two angular positions for trajection of the orthogonal components of linear polarized light and taking two pictures subsequently in both positions of the polarizer filter, in principle. Although this method may be carried out with the accessories of the Zeiss LSM 410, it does not provide satisfying results because of the variation of the intensity of fluorescence in time—especially in biological samples. A further problem may be the variation of the intensity of the illuminating laser light. Vibrations and movements of the sample or the stage may also lead to significant distortion.
U.S. Pat. No. 5,457,536 suggests an improvement to Zeiss LSM which makes the general purpose laser scanning microscope capable of point-by-point measuring the dichroism and the birefringence of the light transmitted through the sample. The laser beam directed to the sample is modulated with a polarization state generator interposed between the laser light source and the sample. For measuring the light transmitted through the sample there is a polarization analyzer on the other side of the object plane. The output of the analyzer is connected to a photodetector which is connected with an output to a demodulator unit. One drawback of this configuration is that in most of the LSM-s the confocal mode is not available during LD, CD and birefringence measurements which can only be carried out in transmission mode. This method does not enable the measurement of the polarization content of the emitted or reflected light, thus the measurement of the anisotropy in the linear or circular polarization (r, CPL) of the emission (emitted light). This is a major drawback in studying biological samples where the confocal fluorescence microscopy is widely used. In most of the biological applications important information on the spatial arrangement of the different components can be obtained by following the emission of several chromophores. Each item of this information carries different polarization information which can not be analyzed with said conventional techniques. In many LSM-s it is not possible or it is very difficult to modulate the laser light because of the optical fiber coupling of the laser light. It is also disadvantageous that there is no possibility to characterize in full detail the polarization content of the light and therefore some of the important parameters—assigned to the Mueller matrix elements—can not be determined.
Therefore one object of the invention is to provide a method and apparatus which combine the advantages of laser scanning microscopy and polarimetry with the combination resulting in more measured parameters with a single apparatus, or with different configurations of a single apparatus with special regard to the parameters of emitted fluorescence measured in confocal mode at a single or multiple wavelengths at substantially the same time, or with regard to the possibly most complete analysis of the polarization content of emitted, transmitted or reflected light.
A further object of the invention is to eliminate or compensate the errors resulting from wavelength dependency and the polarization distortion of the optical devices during the measurements.